During the course of evolution, spontaneous mutations arise in the genomes of organisms. Variations in genomic DNA sequences are created continuously at a rate of about 100 new base changes per individual (Kondrashov, 1995; Crow, 1995). These germ-line changes may produce an evolutionary advantage and be retained in the population, or they may be deleterious and ultimately eliminated. In many cases, equilibrium between multiple germline forms of a sequence is established within a population if reproductive ability of individuals containing either polymorphism is not affected. Over time, significant numbers of mutations have accumulated within the human population that may be observed to varying extents in geographically separated groups based upon the presence of common ancestors.
Colorectal cancer is the third most common cancer and the third most common cause of death from cancer for both men and women. Colorectal cancer is responsible for more deaths that are not due primarily to tobacco use than any other type of cancer and inflicts a huge financial burden. Early detection of some human tumors such as uterine cervical cancer has dramatically reduced mortality from this condition (Herzog, 2003). Early detection of colorectal cancer can reasonably be expected to prevent death from this condition by identifying patients at risk for the disease, or those with the disease in an early stage and allow life saving intervention. A validated genetic test for colorectal cancer predisposition will have clinical utility, allowing prevention of cancer mortality through targeted screening programs. There are good reasons to expect that at least some of the genetic risks of common disease is due to common variants—for example, based on evolutionary arguments, and the fact that most human genetic variation is common. Although approximately 20% of colorectal cancers have a familial component with relatives exhibiting a doubling of risk (Carstensen et al., 1996), less than 5% of colorectal cancer is explained by rare, highly penetrant genetic syndromes such as APC and HNPCC (de Leon et al., 1999). Familial colorectal cancer occurring in patterns inconsistent with classical inherited syndromes suggests that variation in genome sequence plays a major role in determining individual risk to colorectal cancer. These genetic causes appear complex due to a variety of reasons such as genetic heterogeneity, incomplete penetrance, phenocopies and variation in exposures to environmental co-factors etc. There is little insight into the genetic or environmental determinants of almost 90% of cases of human colorectal carcinoma (Lynch and de La, 2003).
Although common human genetic variation is limited compared to other species, it remains impractical to discover and test every one of the estimated 10,000,000 common genotype variants (Sachidanandam et al., 2001) as predictors of disease risk. Genotypic complexity is reduced through linkage disequilibrium that exists across long segments of the human genome with restriction in the diversity of haplotypes observed (Daly et al., 2001; Rioux et al., 2001; Liu et al., 2004). That is, single nucleotide polymorphisms found at specific locations within the human genome are inherited in conjunction with nucleotides that can be polymorphic that are physically located near by. In European genomes, allelic association between pairs of markers typically extends over 10-50k, although there is tremendous variability in the magnitude of association observed at any given distance (Clark et al., 1998; Kikuchi et al., 2003; Dunning et al., 2000; Abecasis et al., 2001). Genome-wide data (Gabriel et al., 2002; Reich et al., 2001; Dawson et al., 2002) supports the generality of this description as well as its application across populations. This confirms that measurement of single nucleotide polymotphisms at sites in tight linkage disequilibrium with adjacent genomic regions can provide information about the presence of diversity not just at sites actually measured, but also about large areas of the adjacent genome.
Numerous types of polymorphisms exist and are created when DNA sequences are either inserted or deleted from the genome. Another source of sequence variation results from the presence of repeated sequences in the genome variously termed short tandem repeats (STR), variable number of tandem repeats (VNTR), short sequence repeats (SSR) or microsatellites. These repeats commonly are comprised of 1 to 5 base pairs. Polymorphism occurs due to variation in the number of repeated sequences found at a particular locus.
The most common form of genomic variability are single nucleotide polymorphisms or SNPs. SNPs account for as much as 90% of human DNA polymorphism (Collins et al., 1998). SNPs are single base pair positions in genomic DNA at which different sequence alternatives (genotypes) exist in a population. By common definition, the least frequent allele occurs at least 1% of the time. These nucleotide substitutions may be a transition, which is the substitution of one purine by another purine or the substitution of one pyrimidine by another, or they may be transversions in which a purine is replaced by a pyrimidine or vice versa.
Typically SNPs are observed in about 1 in 1000 base pairs (Wang et al., 1998; Taillon-Miller et al., 1999). The frequency of SNPs varies with the type and location of the change. Specifically, two-thirds of the substitutions involve the CT (GA) type, which may occur due to 5-methylcytosine deamination reactions that occur commonly. SNPs occur at a much higher frequency in non-coding regions than they do in coding regions.
Known environmental risk factors for the development of colorectal cancer include obesity, absence of a vegetable-rich diet and a sedentary life style. Estrogen use in post menopausal women is associated with reduced individual risk for the development of colorectal cancer. The mechanism of risk reduction through the chronic administration of estrogen is unknown and a way of quantifying altered risk associated with estrogen use is not obvious. It is known that expression of the estrogen receptor beta on colorectal tumors is reduced compared to undiseased adjacent tissue. It is not known if this observation has any relevance to the reduced incidence of colorectal cancer in women taking postmenopausal estrogen, nor is it useful in predicting individual risk for the development of colorectal cancer. It is not known if single nucleotide polymorphisms within the estrogen receptor beta can modify the risk of developing colorectal cancer conferred by certain polymorphisms in other risk genes.